Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

Summary Intracerebral hemorrhage usually manifests as strong neuroinflammation and neurological deficits. There is an urgent need to explore effective methods for the treatment of intracerebral hemorrhage. The therapeutic effect and the possible mechanism of induced neural stem cell transplantation in an intracerebral hemorrhage rat model are still unclear. Our results showed that transplantation of induced neural stem cells could improve neurological deficits by inhibiting inflammation in an intracerebral hemorrhage rat model. Additionally, induced neural stem cell treatment could effectively suppress microglial pyroptosis, which might occur through inhibiting the NF-κB signaling pathway. Induced neural stem cells could also regulate the polarization of microglia and promote the transition of microglia from pro-inflammatory phenotypes to anti-inflammatory phenotypes to exert their anti-inflammatory effects. Overall, induced neural stem cells may be a promising tool for the treatment of intracerebral hemorrhage and other neuroinflammatory diseases.


INTRODUCTION
Although the incidence of intracerebral hemorrhage (ICH) is lower than that of cerebral ischemia, its higher mortality and disability rates deserve our attention. 1 ICH usually manifests as a sudden rupture of an artery in the brain and the release of blood compressing the surrounding brain tissue, causing a rapid increase in intracranial pressure, followed by cerebral edema, inflammatory recruitment, oxidative stress, and bloodbrain barrier disruption. 2,3 ICH is often based on hypertension, and the current treatments for ICH mainly include the surgical removal of hematoma, pharmacological hemostasis, and blood pressure reduction. 4 However, the current rates of therapeutic efficacy and patient recovery are not satisfactory. 5 Therefore, there is an urgent need to explore novel and effective methods for the treatment of ICH.
ICH occurrence indicates that primary cerebral injury is already present; therefore, ameliorating secondary cerebral injury and inhibiting its malignant development are significant strategies in the treatment of ICH. After ICH occurs, the thrombin content in the hematoma area increases, and red blood cells rupture, releasing hemoglobin, iron, reactive oxygen species, and other factors, which then trigger inflammatory responses in the injured area. 3,6 Corresponding studies have found that ICH-induced neuroinflammation is closely related to microglia, which are the most important immune cells in the central nervous system 7 and the earliest immune cells to respond in the brain after ICH. 8 Microglia play a dual role in the different developmental stages of ICH. When ICH occurs, microglia are activated to the pro-inflammatory phenotype, releasing various types of pro-inflammatory factors. However, microglia can also shift from the pro-inflammatory phenotype to the anti-inflammatory phenotype, secreting various types of anti-inflammatory factors, such as interleukin-10 (IL-10), interleukin-4 (IL-4), transforming growth factor-b (TGF-b), and arginase-1 (Arg-1), to suppress the inflammatory response, improve the microenvironment, and promote functional recovery after ICH. 9 Therefore, targeting the mediation of microglial function may become a promising way to treat ICH. 10 Exploring the target mechanisms that involve ICH-induced brain injury and repair is necessary to develop a new and effective therapy for ICH. Microglia triggering neuroinflammation are closely associated with The data are expressed as mean G SD, (n = 4). Compared with the pMSCs group, *p < 0.05 as determined by Student's t test. and a few were Iba-1 positive (5.59% G 1.40%) (Figures 2A and 2B). These results indicated that iNSCs may be differentiated into astrocytes, neurons and microglia in vitro, and astrocytes were dominant.
We also tracked the differentiation of transplanted iNSCs in vivo. On the seventh day of ICH, the transplanted iNSCs were labeled by human nuclear (HN) antigen immunostaining at the same time and colabeled with NeuN, GFAP or Iba-1 antibodies to observe whether the transplanted iNSCs could be differentiated in vivo. Interestingly, the median 36.84% (34.29%-39.53%) of HN-labeled iNSCs were GFAP positive, the median 54.05% (52.34%-58.96%) of HN-labeled iNSCs were NeuN positive, and the median 5.88% (5.00%-6.91%) of HN-labeled iNSCs were Iba-1 positive (Figures 2C and 2D). These results indicated that iNSCs might be mainly differentiated into astrocytes and neurons in vivo, and neurons were dominant.   Figure 3D).
To observe the effect of transplanted iNSCs on neurological deficits in ICH rats, we performed a series of behavioral tests on the first, third, seventh, and 14th days after ICH. The results of the forelimb placing test, corner turning test, and modified neurological severity score (mNSS) showed that compared with the ICH group, the iNSCs group had significantly higher forelimb placement success rates and percentages of right turns on Days 7 (p < 0.05) ( Figures 3E and 3F), while the mNSS score was significantly decreased on Days 3, 7, and 14 (p < 0.05) ( Figure 3G).
In addition, the brain morphology from each group on Day 7 after ICH was examined by Hematoxylineosin (HE) and Nissl staining. HE staining showed that the cells in the sham group had regular morphology, uniform distribution, complete cell bodies, and uniform staining. The ICH group showed a large amount of inflammatory cell infiltration, cell swelling, unclear cell membrane boundaries, and disordered cell arrangement. Compared with the ICH group, the iNSCs group showed less cell swelling, less inflammatory cell infiltration, and relatively uniform cell arrangement ( Figure 3H). Nissl staining revealed pyknotic nuclei or the disappearance of Nissl substance in the ICH group. Compared with the ICH group, the cell morphology was relatively regular and stained uniformly in the iNSCs group ( Figure 3I).

Transplanted iNSCs had effects on perihematomal neural cells in ICH rats
We observed the influence of iNSCs on astrocytes, microglia, and neurons around hematomas through the immunofluorescence staining of GFAP, Iba-1, and NeuN on the seventh day after ICH. Compared with those in the sham group, the numbers of GFAP-positive astrocytes and Iba-1-positive microglia around hematomas in the ICH and iNSCs groups were significantly increased (p < 0.05) ( Figures 4A, 4B, 4D, and 4E). The numbers of astrocytes and microglia around hematomas in the iNSCs group were significantly lower than those in the ICH group (p < 0.05) ( Figures 4A, 4B, 4D, and 4E). Simultaneously, the number of NeuN-positive neurons around hematomas in the ICH and iNSCs groups was significantly decreased compared with that in the sham group (p < 0.05; Figures 4C and 4F). The number of NeuN-positive neurons around hematomas in the iNSCs group was significantly higher than that in the ICH group (p < 0.05) ( Figures 4C and 4F).

iNSCs alleviated inflammation in ICH rats
ICH can cause a series of inflammation, oxidative stress, and other reactions, among which inflammation is dominant. 23 Therefore, we examined the expression of the pro-inflammatory factors CD68, IL-6, and TNFa, as well as the anti-inflammatory factor IL-37 in each group. ICH induced increases in the expression levels of CD68, IL-6, TNF-a, and IL-37, and interestingly, iNSCs transplantation suppressed the expression of CD68, IL-6, and TNF-a, and increased the expression of IL-37 (p < 0.05) ( Figures 5A-5E). These results indicated that iNSCs may inhibit the expansion of inflammation in ICH rats.  iScience Article iNSCs alleviated pyroptosis in ICH rats As pyroptosis plays an important role in the development of inflammation, we detected the expression of pyroptosis pathway-related proteins in each group after 7 days of ICH. The results showed that ICH increased the expression of the nuclear factor-kB (NF-kB) p-P65, NLRP3, apoptosis-associated specklike protein containing (ASC), and pro-Caspase-1, which are used to assemble the NLRP3 inflammasome The Mann-Whitney test in nonparametric test was used to analyze the differences among the groups, and compared with the Sham group *p < 0.05; compared with the ICH group # p < 0.05.

Cell type screening of ICH-induced pyroptosis
To determine which type of neural cells mainly underwent pyroptosis after ICH, we examined the coexpression of neural cell-specific markers and the pyroptosis-related factors NLRP3/Caspase-1 by immunofluorescence double staining.
Based on the above results, we found that pyroptosis mainly occurred in microglia and neurons after ICH and was dominant in microglia. Therefore, we further focused on microglia in our subsequent in vitro experiments. iScience Article

Pyroptosis in microglia was induced in vitro
We stimulated microglia with lipopolysaccharide (LPS)/adenosine-triphosphate (ATP) and triggered their corresponding inflammation and pyroptosis. Iba-1 is the marker protein of microglia, and its expression level increases when microglia are activated by inflammation. 24 CD68 is a marker protein of the pro-inflammatory type after microglial activation. 10 We treated microglia with different concentrations of LPS for 24 h and subsequently treated them with 5.0 mM ATP for 30 min and then observed the expression levels of The Mann-Whitney test in nonparametric test was used to analyze the differences among the groups, and compared with the Sham group *p < 0.05; compared with the ICH group # p < 0.05.

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Iba-1 and CD68. The expression levels of Iba-1 and CD68 showed LPS concentration-dependent trends (p < 0.05), which were not significant when the concentration was over 1.0 mg/mL. Therefore, we selected 1.0 mg/mL as the concentration of LPS for the next step ( Figures 10A, 10D, and 10E).
Inflammation can cause an increase in NLRP3 to trigger pyroptosis. MCC950 is a selective inhibitor of NLRP3. 25 We treated microglia with MCC950 when the cells were incubated with LPS and ATP. The results  iScience Article showed that 2.0 mM MCC950 could effectively inhibit the LPS/ATP-induced increase in NLRP3 (p < 0.05) ( Figures 10B and 10F).
In addition, we explored whether iNSCs affect the pyroptosis of microglia by intervening in the NF-kB signaling pathway. We selected Bay11-7082, which is an inhibitor of NF-kB, 26 and screened the appropriate iScience Article concentration of Bay11-7082. Finally, we selected Bay11-7082 at a concentration of 10 mM for further use because it could inhibit NF-kB p-P65, effectively (p < 0.05) ( Figures 10C and 10G).
Next, we treated LPS/ATP-stimulated microglia with MCC950 or Bay11-7082 or pMSCs or iNSCs by a coculture system and performed staining or extracted proteins after 24 h ( Figure 10H).
iNSCs inhibited LPS/ATP-induced pyroptosis in microglia NLRP3 expression in microglia in vitro We performed Iba-1/NLRP3 immunofluorescence double staining on microglia in each group to observe pyroptosis in microglia. The percentages of NLRP3-labeled Iba-1-positive cells were 5.69% G 1.57% in the control group, 61.97% G 4.11% in the LPS/ATP group, 33.27% G 4.32% in the MCC950 group, 26.22% G 5.04% in the pMSCs group, and 18.07% G 3.87% in the iNSCs group. Compared with that in the control group, the number of NLRP3-positive microglia was significantly increased in the LPS/ATP group (p < 0.05). Moreover, compared with that in the LPS/ATP group, the numbers of NLRP3-positive microglia in the MCC950, pMSCs, and iNSCs groups were significantly decreased (p < 0.05). These results indicated that MCC950, pMSCs, and iNSCs could reduce the level of microglial pyroptosis. Compared with that in the pMSCs group, the number of NLRP3-positive cells in the iNSCs group decreased significantly (p < 0.05) ( Figures 11A and 11B).

Caspase-1 expression in microglia in vitro
Similarly, we performed Iba-1/Caspase-1 immunofluorescence costaining on microglia in each group to examine pyroptosis in microglia. The percentages of Caspase-1-labeled Iba-1-positive cells were 6.33% G 1.22% in the control group, 58.95% G 5.02% in the LPS/ATP group, 26.30% G 3.77% in the MCC950 group, 19.80% G 3.12% in the pMSCs group, and 12.72% G 2.42% in the iNSCs group. Compared with that in the control group, the number of Caspase-1-positive microglia was significantly increased in the LPS/ATP group (p < 0.05). However, the numbers of Caspase-1-positive cells in the MCC950, pMSCs, and iNSCs groups were significantly decreased compared with that in the LPS/ATP group (p < 0.05). Compared with the pMSCs group, the number of Caspase-1-positive cells in the iNSCs group was significantly decreased (p < 0.05) ( Figures 12A and 12B).
All the above results indicated that iNSCs had more advantages in the inhibition of microglial pyroptosis induced by LPS/ATP than pMSCs and MCC950.

Metabolomic analysis of microglia: Clues for future studies
To further clarify the possible mechanisms of iNSCs in microglial pyroptosis, we performed metabolomic assays on microglia from each group. The KEGG pathway enrichment results showed that MCC950, pMSCs, and iNSCs were involved in regulating the inflammation-related cAMP signaling pathway, the alanine, aspartate, and glutamate metabolism pathway, and the antioxidant-related pentose phosphate pathway ( Figures 15A, 15B, and 15C). There were 29 differential metabolites coexpressed in the MCC950, pMSCs, and iNSCs groups ( Figures 15D and 15E). The heatmap constructed from the results revealed that after treatment with MCC950, pMSCs or iNSCs, the abundance of ascorbic acid, which has antioxidant and anti-inflammatory effects, 27 significantly increased. Meanwhile, the abundance of 2-hydroxyethyl methacrylate, which promotes inflammation, 28 significantly decreased ( Figure 15E). The volcano plot results showed a higher log 10 p-value of ascorbic acid in microglia after iNSCs treatment, and the expression levels of spermine and quinolizidine, which inhibit inflammation 29,30 were significantly increased only in the iNSCs group ( Figures 15F, 15G, and 15H).

DISCUSSION
NSCs have been considered to possess valuable potential to treat cerebral diseases for which present therapies are almost unavailable due to their ability to differentiate into neurons and glial cells or their paracrine mechanisms. 31,32 Because the original source of NSCs is deficient, many studies have challenged the treatment of nervous diseases with induced NSCs from different types of stem cells, 33 but studies on the effects and mechanisms of MSC-induced NSCs on ICH treatment are rare. In this study, pMSCs were successfully induced into the spheroidal morphology of NSCs. The iNSCs highly expressed Nestin and Sox-2, markers of NSCs, 34 and secreted a series of neurotrophic factors, 35 which can promote nerve development, repair nerve damage, and regulate nervous system homeostasis. Although, the iNSCs showed multidirectional differentiation ability, they tended to differentiate into astrocytes in vitro while mainly differentiating into neurons in vivo. The microenvironments of iNSCs in vitro were different from those in vivo, which likely explains why the iNSCs exhibited their different main differentiation directions.
After discovering that transplantation of iNSCs could improve ICH-induced neurological deficit symptoms, we searched for reasonable mechanisms for these results. ICH is an exceedingly destructive cerebrovascular disease. Once ICH occurs, a large number of cells die in the brain tissue, resulting in neurological dysfunction. Our results showed that the transplanted iNSCs could be differentiated into neuron-and glia-like cells. To determine whether these differentiated cells can replace the function of the dead neuronal cells, the ''real'' functions of neuronal cells, such as synaptic transmission, maintenance of a resting membrane potential, and the ability of fire trains of action potentials will need to be tested in future studies. 36 In this study, we mainly focused on the paracrine mechanisms of iNSCs, as a number of neurotrophic factors are secreted from iNSCs.
It is well-known that inflammation plays an important role during the acute phase of hemorrhagic stroke, which leads to secondary brain injury. 37 Many studies have tried to inhibit ICH-induced inflammation through different treatments. 38 Our results demonstrated that iNSCs transplantation could effectively suppress ICH-induced inflammation. Microglia are vulnerable to ICH, and their activation  iScience Article can be divided into pro-inflammatory and anti-inflammatory phenotypes. Microglia of the pro-inflammatory phenotype mainly express CD68, CD86, and iNOS, and microglia of the anti-inflammatory phenotype mainly express CD206, Arg-1, and other factors. 10 Under certain conditions, pro-inflammatory and anti-inflammatory phenotypes can be mutually transformed. 39 Therefore, scholars have recently investigated how to induce the conversion of microglia from the pro-inflammatory to the antiinflammatory phenotype to exert anti-inflammatory effects. Zhou, 40 Miao, 41 and Tian 42 et al. found that inducing the conversion of microglia from pro-inflammatory to anti-inflammatory could improve inflammatory damage in ICH rats. Our results also showed that iNSCs treatment could reduce the protein expression levels of the microglial pro-inflammatory phenotype markers CD68 and iNOS and the pro-inflammatory factor IL-6 while upregulating the expression of the microglial anti-inflammatory phenotype marker Arg-1 and the antioxidant factor SOD. Our results indicated that iNSCs treatment could promote the conversion of activated microglia from the pro-inflammatory to the anti-inflammatory phenotype and exert anti-inflammatory effects. Simultaneously, we compared the anti-inflammatory effect of iNSCs with those of pMSCs, MCC950 or Bay 11-7082, which have anti-inflammatory effects, 26,43,44 and the results indicated that iNSCs were more advantageous in the treatment of neuroinflammationrelated diseases.
Previous studies have proven that pyroptosis is the key mechanism of ICH-induced inflammation. 11 The specific process of pyroptosis consists of two parts: initiation and activation. In the initiation process, when ICH occurs, a series of stimuli, such as hemoglobin, thrombin, and reactive oxygen species cause an upregulation of NLRP3 expression in microglia, which in turn recruits ASC and pro-Caspase-1 to form the NLRP3 inflammasome, 45 and NF-kB translocates to the nucleus to enhance the transcription levels of NLRP3, pro-IL-1b, and pro-IL-18 genes. 46 In addition, the activation process involves the NLRP3 inflammasome, which activates pro-Caspase-1 to form mature Caspase-1, 47 and Caspase-1, which further cleaves pro-IL-1b and pro-IL-18 to form mature IL-1b and IL-18. 48 Moreover, Caspase-1 can also cleave GSDMD on the membrane to form the GSDMD N-terminus, forming a membrane pore that leads to the release of extracellular contents, such as the inflammatory factors IL-1b and IL-18, resulting in the further infiltration of inflammatory cells and exacerbating the inflammatory response. 49 Fortunately, our results proved that pyroptosis occurred in the ICH model and that iNSCs transplantation inhibited the expression of active NF-kB, the NLRP3 inflammasome, active Caspase-1, IL-1b and IL-18, all of which are typical markers of pyroptosis-related inflammation. These results implied that iNSCs transplantation could suppress inflammation by inhibiting the NF-kB-mediated pyroptosis pathway.
Previous studies have shown that the formation of the NLRP3 inflammasome is mainly reflected in microglia after ICH occurs. 46,50 Our results also showed that ICH mainly caused microglial pyroptosis. Therefore, we selected microglial cells to verify the detailed therapeutic mechanisms of iNSCs on microglial pyroptosis in vitro. MCC950 is a selective inhibitor of NLRP3 and has therapeutic effects on NLRP3-related inflammatory or immune diseases. 25 Ren, 51 Wang, 52 and Xiao 53 et al. found that MCC950 could reduce the degrees of nerve damage and inflammation after ICH. In addition, Liu 43 and Chivero 54 et al. found that MCC950 can inhibit microglial activation and reduce neuroinflammation. Our results indicated that both MCC950 and iNSCs could inhibit microglial NLRP3 upregulation. Nevertheless, there are many parameters, such as TNF-a, IL-6, pro-Caspase-1, Caspase-1 P10 and Caspase-1 P20, especially NF-kB p-P65, were effectively suppressed by iNSCs or iNSCs combined with MCC950, but were not affected or only modestly affected by MCC950. That means, the effect of iNSCs may be dependent on the mechanisms other than NLRP3.
The activity of NF-kB is a key regulatory factor of pyroptosis, and Bay 11-7082 is an inhibitor of the NF-kB signaling pathway, which has long been regarded as a typical pro-inflammatory signal transduction pathway. 55 Surprisingly, iNSCs treatment suppressed the activation of NF-kB, and iNSCs combined with Bay 11-7082 treatment showed a greater inhibitory effect on NF-kB activation than iNSCs or Bay 11-7082 treatment alone. Therefore, we speculated that iNSCs might inhibit microglial pyroptosis by inhibiting the NF-kB signaling pathway. (D) Venn diagram of the differential metabolites between the MCC950, pMSCs, and iNSCs groups and the LPS/ATP group, respectively. (E) Heatmap of 29 differential metabolites co-expressed between the MCC950, pMSCs, and iNSCs groups compared with the LPS/ATP group, respectively. (F-H) Volcano plots of differential metabolites between the MCC950, pMSCs, and iNSCs groups compared with the LPS/ATP group, respectively. The red and blue dots indicated up-regulated and down-regulated differential metabolites, respectively (VIP >1, p < 0.05).

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iScience 26, 107022, July 21, 2023 21 iScience Article It was impossible to clarify the single target on which the iNSCs act during their treatment due to the cells' secretion of multiple cytokines and chemokines. However, we wanted to determine other changes in the microenvironment after iNSCs treatment. Cell metabolism is a basic life activity by which cells maintain their own energy balance and nutrient supply. Inflammation is usually accompanied by changes in cellular metabolism, 56 which can also further affect the progression of inflammation. 57 Our metabolomic results showed that MCC950, pMSCs, and iNSCs were all involved in regulating the inflammation-related cAMP signaling pathway, 58 alanine, aspartate and glutamate metabolism, 59 and the antioxidant-related pentose phosphate pathway. 60 Next, we focused on 2-hydroxyethyl methacrylate and ascorbic acid, which are related to inflammation and oxidative stress. Sara et al. 28 found that 2-hydroxyethyl methacrylate was closely associated with NLRP3 inflammasome activation and that 2-hydroxyethyl methacrylate had a proinflammatory effect. Ascorbic acid plays a significant role in inhibiting apoptosis and antioxidation. 61 We further observed the expression of spermine and quinolizidine in each group. Xu et al. found that spermine attenuated inflammation and apoptosis in the aging brain. 29 Quinolizidine alkaloids have anti-inflammatory, antiviral, antioxidant, and neuroprotective effects. 30,62 Our results indicated that iNSCs treatment might cause the inflammatory environment to change by regulating the metabolism of 2-hydroxyethyl methacrylate, ascorbic acid, spermine and quinolizidine alkaloids in microglia. We will perform further studies to verify this possibility.
Taken together, our results demonstrated that iNSCs transplantation could improve neurological deficits in ICH rats by inhibiting ICH-induced inflammation. Additionally, iNSCs treatment could effectively suppress microglial pyroptosis, which might occur through inhibiting the NF-kB signaling pathway. Furthermore, iNSCs could regulate the polarization of microglia and promote the transition of microglia from a pro-inflammatory to an anti-inflammatory phenotype to exert anti-inflammatory effects. Overall, iNSCs may be a promising stem cell species for the treatment of ICH and other neuroinflammatory diseases in the future.

Limitations of the study
One limitation of this study is that we found that the transplanted iNSCs could be differentiated into neuron-and glia-like cells. Regarding whether these differentiated cells could replace the functions of the dead neuronal cells, we have not tested the ''real'' functions of neuronal cells, such as synaptic transmission, maintenance of a resting membrane potential, and the ability of fire trains of action potentials; these possibilities will be further examined in future studies.
Second, there are multiple approaches to establish ICH models. Examples include autologous blood injection, collagenase injection, blood component injection, and laser-induced rupture of vessels. Because the etiology of spontaneous ICH is very complex, there is currently no method of ICH modeling that can fully reflect the characteristics of human clinical ICH. 63 Therefore, in our future research, we will also improve our model methods and incorporate multiple complications (including hypertension, dyslipidemia, and diabetes) into an ICH model to better reflect the actual situation of ICH in clinical scenarios.
In addition, some commonly used behavioral tests, including mNSS, can be affected by other factors, such as severe weight loss in animals, infection and other complications. 64 Therefore, more rigorous behavioral tests, such as tapered/ledged beam walking, the cylinder test, and Montoya's staircase, will be incorporated into future studies to further improve the experimental design.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

pMSCs cell culture
The pMSCs used in this experiment were provided by the Ningxia Key Laboratory of Stem Cells and Regenerative Medicine, and the collection and acquisition of the cells were approved by the Ethics Committee of the General Hospital of Ningxia Medical University. The pMSCs were derived from human placental tissues, which were provided by four different donors, and extracted according to our previous research methods. 65 The pMSCs expressed the mesenchymal stem cell surface markers CD73, CD90 and CD105 but did not express CD14, CD45, CD34 and HLA-DR (data not show). The pMSCs also showed strong proliferation ability in vitro. The pMSCs were cultured in serum-free Ultra Culture Serum-free Medium (Lonza, Switzerland) supplemented with 2% Pall Ultroser Gä Serum Substitute (Pall, USA) at 37 C in a 5% CO 2 incubator. . When the cultured pMSC density reached 80%-90%, the cells were digested with TrypLEä (1x, Gibco, USA), and 2310 6 pMSCs were cultured with iNSCs induction medium in a 100 mm culture dish at 37 C in a 5% CO 2 incubator. Neurospheres formation were observed by Olympus (Olympus, Japan) phase-contrast microscopy on the 1st, 3rd and 7th days. Subsequently, 3-4 ml of fresh iNSCs induction medium was used to replenish the culture dish every 3 days. After 10 days of induction culture, a portion of the neurospheres were used for further differentiation culture, and the other portion was used for transplantation to the ICH rats or cocultured with microglia.

iNSCs differentiation culture
The neurospheres were digested with TrypLEä for 5 minutes and blown gently to separate them into single cells. Then, the single cells were cultured with differentiation medium containing the following components: 97% DMEM/F12, 2% FBS and 1% N-2 supplement. After 7 days of culture, the differentiation efficiency of iNSCs was detected by immunofluorescence staining.

ICH model establishment and iNSCs transplantation
The experimental animals were randomly numbered and grouped using the random number table method. The experimental animals were randomly divided into the sham group (n=21), ICH group (n=21), and iNSCs group (n=21). The environment and surgical instruments were sterilized before surgery. The rats were anesthetized with isoflurane, and 2 ml of 0.2 U type VII collagenase (C2399, Sigma-Aldrich) was injected into the left striatum with a microinjector at a controlled rate of 0.4 ml/min. The stereotaxic coordinates for collagenase type VII injection were as follows: anteroposterior (AP) -0.2 mm, mediolateral (ML) 3.0 mm, dorsoventral (DV) 6.0 mm (left). After the injection, the needle was stopped for 10 min, the syringe was slowly withdrawn vertically, the needle hole was closed with bone wax, and the wound was sutured. The sham group underwent the same operation without collagenase type VII injection. iScience Article For the iNSCs group, twenty-four hours after ICH induction, the iNSCs were collected and pretreated with TrypLEä for 5 min, and the neurospheres were gently blown to separate them into single cells. The ICH model rats were anesthetized, and 30 ml of 1310 6 iNSCs were slowly injected into the same position as collagenase type VII injection. The sham and ICH groups were positionally injected with 30 ml PBS, and the other procedures were similar to those of the iNSCs group.
In addition, the following measures were used to alleviate pain during the operation on the experimental animals.
(1) The surgical environment and surgical instruments were fully disinfected before the operation to reduce postoperative infection.
(2) Isoflurane anesthesia with a high safety factor was used to avoid the pain of subcutaneous anesthetic injection, with the anesthetic concentration adjusted according to the experimental needs to reduce pain caused by skin suture and other irritations in the rats. (3) After the operation, the rats were placed on a body heat plate and transferred to their cages after waking up to avoid hypothermia. (4) The rats were given carprofen (5 mg/kg) for analgesia before recovery from anesthesia and once every 24 hours for 72 h after surgery. The animals were also checked daily for pain, discomfort and wounds. (5) All the animals were sacrificed separately to prevent the other rats from suffering from fear and mental pain.

Tissue immunofluorescence staining
Frozen sections of rat brain tissue with a thickness of 10 mm were harvested from each group and washed three times with PBS, treated with 0.5% Triton X 100 for 20 min, and blocked with 5% goat serum at room temperature for 1 h. The sections were incubated with the following primary antibodies overnight at 4 C: NeuN (1:200, ab104225, abcam), GFAP (1:300, ab7260, abcam), Iba-1 (1:500, 019-19741, Wako), NLRP3 (1:200, YT5382, Immunoway), and Caspase-1 (1:300, YT5743, Immunoway). The following day, the sections were incubated with the fluorescence-labeled secondary antibodies Alexa Fluorâ 555 or Alexa Fluorâ 488 for 2 h at room temperature in the dark. DAPI staining solution was added to the sections and observed under a fluorescence microscope (Olympus, Japan), and images were acquired. For analysis of the staining results, five samples from each group were selected, three sections were selected from each sample, five areas around the hematoma were randomly photographed in each section, and the number of positive cells was counted.

Hematoma volume quantification
On the 7th day, the rat brain tissue of each group was collected and sliced to observe the size of the hematoma. The rat brain tissue was cut into ten consecutive 1-mm-thick tissue sections, and the hematoma size of brain tissue in each group was quantified by ImageJ. Hematoma volume rate = hematoma volume/ whole brain volume 3 100%.

Brain water content assay
The wet and dry specific gravity method was used to assess the degree of brain edema after ICH. On the 7th day, each group of rats was anesthetized separately, brain tissue was quickly removed, and the cerebellum and brain stem were discarded, separated into two hemispheres along the midline and divided into the injured side and the contralateral side. The wet weight of the brain tissue on the injured side of each group was determined with an electronic analytical balance and recorded. Then, the brain tissue was wrapped in ll OPEN ACCESS

Neurobehavioral assessment
(1) The forelimb placing test was used to evaluate the sensorimotor impairment of animals. Before the experiment, the rat was given the opportunity to relax and gently grasped to suspend its limbs, after which the rat's right whiskers were allowed touch the corner of a table to induce it to place a forelimb on the table. For ICH rats, contralateral forelimb placement presented a certain obstacle, and each rat was tested 10 times. The number of times the rat's right forelimb was placed was recorded, and the score was calculated as follows: score = number of right forelimb placements/total number 3 100%. A higher score indicated that the animal's injury was relatively minor.
(2) The corner turning test was used to evaluate the damage to the animal's limb coordination function. The specific method involved placing the rat in a 30-degree angle device formed by two baffles. The animal was then turned around, and each rat was tested 10 times. The number of times the rat turned right was recorded. Each test interval was greater than 30 s, and the score was calculated as follows: score = number of right turns/total number of times 3 100%. The higher the score was, the more minor the damage to the animal.
(3) The Modified Neurological Severity Score (mNSS) was used to reflect the neurological deficit of experimental animals. This score mainly included the evaluation of the animal's motor, sensory, reflex, balance, muscle strength and other functions. The score was quantified as 0-18 points; the higher the score was, the more serious the neurological deficit.
Hematoxylin and eosin (HE) staining and Nissl staining HE staining was used to observe the morphology, quantity and distribution of normal and diseased cells around the brain tissue. Nissl staining was used to observe the size and number of Nissl bodies in the cytoplasm and dendrites of neurons. According to the instructions of the HE staining kit (Solarbio, China) and the Nissl staining kit (Solarbio, China), the frozen sections of each group were stained as indicated, and images were collected under an upright microscope (Olympus, Japan).
In vitro microglia pyroptosis model The experiment was divided into 8 groups. Control group: Microglia were cultured with complete medium (DMEM+10% FBS). LPS/ATP group: Microglia (2310 5 /well) were seeded in a 6-well plate. After microglia adhered to the wall the next day, the culture medium in the well was discarded, followed by stimulation with 1.0 mg/ml lipopolysaccharide (LPS; Sigma-Aldrich, USA) for 24 h and subsequent treatment with 5.0 mM adenosine-triphosphate (ATP; Sigma-Aldrich, USA) for 30 min to induce microglia pyroptosis, after which the medium was replaced with normal complete medium and culturing for 24 h. MCC950 and Bay 11-7082 groups: The treatment steps were the same as in the LPS/ATP group, except that complete medium with a concentration of 2.0 mM MCC950 (HY-12815A, MCE, USA) or 10.0 mM Bay 11-7082 (HY-13453, MCE, USA) was used for the replacement, after which the cells were cultured for 24 h. The pMSCs and iNSCs groups were treated with the same steps as the LPS/ATP group, except that 3310 5 /well pMSCs or 300/well iNSCs (diameter: 100 mm-200 mm) were added to the Transwell plate inserts (Corning, #3450, USA) and cocultured with microglia for 24 h. The iNSCs+MCC950 and iNSCs+Bay 11-7082 groups were also treated with the same steps as the iNSCs group, except that complete medium with a concentration of 2.0 mM MCC950 or 10.0 mM Bay 11-7082 was used for the replacement, after which the cells were cultured for another 24 h. The microglial proteins were extracted for western blot detection. The supernatants from the control, LPS/ATP, MCC950, pMSCs and iNSCs groups were then collected for ELISA detection.

Western blot analysis
On the 7th day, the rats in each group were anesthetized, the brain tissue was quickly removed, and the front and rear of the injection site were expanded by 1 mm based on the coronal plane to obtain brain tissue slices with a thickness of 2 mm. The samples were quickly frozen in liquid nitrogen and then transferred to a -80 C freezer for storage. IP Lysis buffer (Cat. No: 87787; Thermo) with protease and phosphatase inhibitors (Cat. No: 78441; Thermo) was added to the separated brain tissues or cells, which were then ground in a tissue grinder for 30 min. The supernatants were collected by centrifugation at 12000 r/min for 15